1. Field of the Invention
The field of this invention relates to methods of controlling the tumorigenicity and/or metastasis of neoplastic cells. Specifically it relates to the use of the R1 gene sequence of ribonucleotide reductase and gene product thereof to suppress malignancy.
2. Description of Related Art
The first unique step leading to DNA synthesis is the conversion of ribonucleotides to their corresponding deoxyribonucleotides, a reaction that is catalyzed in a cell cycle specific manner by the housekeeping gene ribonucleotide reductase [Lewis et al., 1978; Reichard, 1993; Wright, 1989a; Wright et al., 1990a; Stubbe, 1989]. The mammalian enzyme is composed of two dissimilar dimeric protein components often called R1 and R2, which are encoded by two different genes located on different chromosomes [Bjxc3x6rklund et al., 1993; Tonin et al., 1987]. Mammalian protein R1 is a homodimeric structure and has substrate sites and allosteric effector sites that control enzyme activity and substrate specificity [Wright, 1989b; Thelander et al., 1980; Caras et al., 1985; Wright et al., 1990a]. Protein R2 is a homodimer and forms two equivalent dinuclear iron centers that stabilizes a tyrosyl free radical required for catalysis [Wright et al., 1990a; Thelander et al., 1985; McClarty et al., 1990]. R1 and R2 proteins interact at their C-terminal ends to form an active holoenzyme [Reichard, 1993; Wright et al., 1990a; Davis et al., 1994]. Ribonucleotide reductase serves other biological functions in addition to providing substrates for DNA replication. For example, its activity can be induced outside the S phase by DNA cross-linking agents such as chlorambucil and UV irradiation, indicating a role for the enzyme in the DNA repair process [Hurta and Wright, 1992].
R1 and R2 are differentially regulated during the cell cycle. There is an S-phase correlated increase in the R2 protein resulting from its de novo synthesis [Lewis et al., 1978; Mann et al, 1988]. The activity of ribonucleotide reductase, and therefore DNA synthesis and cell proliferation, is controlled in proliferating cells during the cell cycle by the synthesis and degradation of the R2 component [Eriksson et al., 1984; Choy et al, 1988]. The rate-limiting R2 component is a phosphoprotein capable of being phosphorylated by the CDC2 and CDK2 protein kinase mediators of cell cycle progression [Chan et al., 1993], and contains non-heme iron that stabilizes an unique tyrosyl free radical required for enzyme activity [Reichard, 1993; McClarty et al., 1990].
The levels of the R1 protein do not appear to change substantially during the cell cycle of proliferating cells and can be detected throughout the cell cycle. Synthesis of R1 mRNA, like R2 mRNA appears to occur mainly during S phase [Eriksson et al., 1984; Choy et al., 1988; Mann et al., 1988]. The broader distribution of the R1 protein during the cell cycle is attributed to its longer half life as compared to the R2 protein [Choy et al., 1988; Mann et al., 1988].
Regulation of ribonucleotide reductase, and particularly the R2 component, is markedly altered in malignant cells exposed to tumor promoters or to the growth factor TGF-xcex2 [Amara, et al., 1994; Chen et al., 1993; Amara et al., 1995b; Hurta and Wright, 1995; Hurta et al., 1991]. An R1 deletion can be detected in some human colorectal carcinomas [Glenney, 1986]. Higher levels of enzyme activity have been observed in cultured malignant cells when compared to nonmalignant cells [Weber, 1983; Takeda and Weber, 1981; Wright et al., 1989a], and increased levels of R2 protein and R2 mRNA have been found in pre-malignant and malignant tissues as compared to normal control tissue samples [Saeki et al., 1995; Jensen et al., 1994]. Regulation of ribonucleotide reductase, and in particular the R2 component, is significantly elevated in transformed cells exposed to tumor promoters, or to transforming growth factor xcex2 in growth factor mediated mechanisms of tumor progression [Amara et al., 1996; Chen et al., 1993; Amara et al, 1995b].
Currently chemotherapeutic compounds like hydroxyurea inhibit ribonucleotide reductase activity by destabilizing the iron center of the R2 protein causing the destruction of the tyrosyl free radical [McClarty et al., 1990], and preventing cells from progressing through S-phase of the cell cycle [Ashihara and Baserga, 1979]. Such drugs have a limited usefulness in treatment of human cancer and therefore additional approaches which target ribonucleotide reductase are needed.
Breakthroughs in molecular biology and the human genome project have opened previously unforeseen possibilities for targeted intervention with mammalian gene expression [Blaese, 1997; Felgner, 1997]. These include approaches such as gene therapy to introduce into neoplastic cells genetic control sequences and specific proteins to kill proliferating cells. It would be useful to utilize this approach to modify expression of ribonucleotide reductase in cells in which growth must be controlled such as a neoplastic cells.
According to the present invention, a use of a growth modulating amount of an expressible nucleic acid sequence for ribonucleotide reductase R1 to modulate the tumorigenic and metastatic properties of a cell in a human or other mammal is disclosed. The present invention also provides a method of modulating the tumorigenic and metastatic properties of a cell in a human or other mammal. The method includes the steps of contacting a neoplastic cell with a growth modulating amount of an expressible nucleic acid sequence for ribonucleotide reductase R1 of the mammal and in an embodiment for humans can be SEQ ID No:1 or the specific R1 coding sequence thereof. The expressible nucleic acid sequence can be delivered via a gene delivery vehicle which can be in the form of a vector for gene therapy. Alternatively in an embodiment the R1 gene product protein or biologically active peptide thereof which can be in the form of a pharmaceutical composition can be delivered to the cell to be controlled. The method and use and composition of the present invention provides for a generally elevated expression of the R1 component of mammalian ribonucleotide reductase in a cell to be controlled.